Protective Mechanism Study Of Thymoquinone On Lung Injury Induced By PM2.5 In Rats

ObjectiveTo explore the mechanism of PM2.5-induced lung injury in rats and the protective mechanism of Thymoquinone(TQ)on lung injury induced by PM2.5.MethodsForty-two male Wis tar rats were randomly divided into 7 groups(n=6),respectively:group A(blank control group:no treatment),group B:(saline control group:Saline tracheal instillation),group C(TQ control group:saline tracheal instillation+ TQ 40 mg/kg),group D(PM2.5 exposure group:tracheal instillation of 7.5 mg/kg PM2.5),E group(solvent control group:tracheal instillation of 7.5 mg/kg PM2.5+ equal volume of TQ solvent),group F(low dose TQ group:tracheal instillation of 7.5 mg/kg PM2.5+ TQ 20 mg/kg)and group G(high-dose TQ group:tracheal instillation of 7.5 mg/kg PM2.5+TQ 40 mg/kg).Each group of PM2.5 and saline was instilled once a day for 14 days.Each group of TQ gavage should be given 20 mg/kg or 40 mg/kg one day before the experiment,once a day until the end of the experiment.The rats were sacrificed 24 hours after the last PM2.5 exposure and TQ gavage.The bronchoalveolar lavage fluid(BALF)of each group was collected,and the cell count and lactate dehydrogenase(LDH)and total protein(TP)in the supernatant were collected.The levels of interleukin-1?(IL-1?),interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?)were measured.The lung tissues of each group were collected,and the pathological changes of lung tissue and pathological scores were observed by HE staining.The mRNA expression of inflammatory factors IL-1?,IL-6 and TNF-a in lung tissue was determined by PCR.The expression of oxidation and antioxidant indicators MDA,SOD,GSH-PX,Cytoplasmic Nr2,Nuclear Nrf2 and HO-1 Protein were determined according to the kit or Western blotting.The expression of autophagy marker proteins LC3?/? and Beclinl was detected by Western blotting and the apoptosis of lung tissue was detected by TUNEL staining.In addition,the protein expression levels of TNF-?,IL-6,NF-?B p65(cytoplasm,nucleus),HO-1 and ET-1 were detected by Western blotting in groups A,B and D.Analysis and mapping were performed using SPSS 22.0 and Graphpad prism 5.0.All measurement data were expressed as mean±standard deviation(X±S).Cell count statistics were analyzed by Kruskal-Wallis test in non-parametric test and one-way ANOVA was used for the comparison of the mean values of the other groups.If the variance was homogeneous,Tukey test was used;if the variance was not homogeneous,games-howell test was used.The test level =0.05.Results1.General signs of experimental rats:all rats survived during the experiment.Compared with group A and B,rats in the PM2.5 exposure group had poorer mental state,less rapid response to external stimuli,faster breathing,increased oral and nasal secretions,reduced activity,dry and dull fur,and slower weight gain.The symptoms of rats exposed to the low-and high-dose of TQ were similar to those of rats exposed to PM2.5,but the severity was less.2.Pathological examination showed that,compared with group A and B,the alveolar junction of rats exposed to PM2.5 was broken,red blood cell exudation and particulate matter deposition were observed in the alveolar cavity and interstitium,and the pulmonary interstitium was significantly broadened,edematous and infiltrated by a large number of inflammatory cells.The pathological scores were higher,there was a statistical difference(P<0.05).Compared with the PM2.5 exposure group,the TQ high-dose group showed significant improvement in lung histopathological morphology and lower pathological score(P<0.05),while the TQ low-dose group showed a difference in lung histopathological changes between the two groups.3.Compared with group A and group B,the levels of LDH and TP in BALF of rats exposed to PM2.5 were significantly increased(P<0.01).The levels of TP and LDH in BALF of the TQ high-dose group were significantly lower than those of the PM2.5 exposure group(P<0.01 or P<0.05).4.Compared with group A and B,MDA level,nuclear Nrf2 and HO-1 antioxidant protein expression were increased,and cytoplasmic Nrf2 protein,SOD and GSH-Px antioxidant enzyme levels were decreased in rats exposed to PM2.5,showing statistically significant difference(P<0.01).The cytoplasmic Nrf2 in the lung tissue of the low-and high-dose TQ group was lower than that in the PM2.5 group,and the expression of nuclear Nrf2 and HO-1 was increased in a dose-dependent manner,which was statistically significant(P<0.01).The MDA content in the lung tissue of the TQ high dose group was lower than that of the PM2.5 group(P<0.05),and the GSH-Px antioxidant enzyme content was increased,which was statistically significant(P<0.01).5.Compared with group A and B,the expression levels of cytoplasmic NF-?B protein was decreased in the lung tissues of rats exposed to PM2.5,while the expression of nuclear NF-?B protein,TNF-? mRNA,IL-1? mRNA,IL-6 mRNA,TNF-a protein,IL-6 protein,HO-1 protein,ET-1 protein in the lung tissues and the levels of TNF-?,IL-6,IL-1?,TP and LDH were increased in BALF of rats exposed to PM2.5,showing statistical difference(P<0.01 or P<0.05).The expression of TNF-a mRNA in lung tissue of the low-and high-dose TQ group was decreased in a dose-dependent manner,and the levels of IL-6,TP in BALF were significantly decreased(P<0.01).In addition,the expression of IL-1? mRNA in lung tissue and the levels of TNF-?,IL-1? and LDH in BALF were significantly lower in TQ high dose group than in PM2.5 group(P<0.05).6.Compared with group A or B,the total number of leukocytes,neutrophils,macrophages and lymphocytes in BALF of rats exposed to PM2.5 group were significantly increased(P<0.01).The low and high dose groups of TQ showed a tendency to reduce inflammatory cells.7.Compared with group A and B,TUNEL staining of rats exposed to PM2.5 showed a large number of yellow or brown-yellow positive cells in bronchial epithelial cells and alveolar epithelial cells.Only a small number of TUNEL-positive cells were found in the lung tissues ofthe TQ high-dose gro up compared with the PM2.5 exposure group,while the TQ low-dose group was varied between the two groups.8.Compared with group A and B,the expressions of LC3?/? and Beclinl proteins labeled with autophagy proteins in lung tissues of rats exposed to PM2.5 were increased,with statistical difference(P<0.01).Compared with PM2.5 exposure gro up,LC3?/? and Beclinl protein expressions in lung tissues of rats in the low-and high-dose TQ groups were decreased in a dose-dependent manner,with statistically significant differences(P<0.01 or P<0.05).Conclusions1.PM2.5 exposure can impaire the vascular endothelium and alveolar epithelial barrier function and induce oxidative and antioxidant imbalances,inflammatory reactions,cell apoptosis and severe cellular autophagy in rat lung tissue,which leads to lung injury.Activation of NF-?B signaling pathway may be one of the main mechanisms of lung injury induced by PM2.5 exposure.2.TQ can antagonize lung injury induced by PM2.5,which mechanism may inhibit NF-?B signaling pathway-mediated inflammation and activate Nrf2/HO-1/GSH-PX antioxidant signaling pathway,and also inhibit cells apoptosis and autophagy,thus protecting the lung tissue.3.TQ has a dose-dependent protective effect on lung injury induced by PM2.5.